Capybara density and climatic factors as modulators of Ehrlichia prevalence in questing ticks in the Iberá wetlands, Argentina

We evaluated the presence of Ehrlichia spp. in unfed capybara ticks, Amblyomma dubitatum, and explored its association with capybaras density, ticks density and environmental variables. We observed that in the Iberá wetlands ecoregion A. dubitatum is infected by "Candidatus Ehrlichia hydrochoerus” and in a lesser extent with an Ehrlichia species closely related to Ehrlichia chaffeensis. The frequency of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum was not associated with vector abundance, but the probability of finding "Ca. Ehrlichia hydrochoerus"-infected ticks increased when the density of capybaras was low two months before. We hypothesize that when the density of capybaras decreases, A. dubitatum immature stages may seek out alternative hosts one of which could exhibit high realized reservoir competence for "Ca. Ehrlichia hydrochoerus", leading to an increased prevalence of this ehrlichiae in questing A. dubitatum. High minimum temperatures and high cumulative rainfall in the time period previous to tick collection (15 to 60 days) were positively correlated with the prevalence of "Ca. Ehrlichia hydrochoerus" infection in A. dubitatum. Our results suggest that a combination of factors (both biological and abiotic) could raise the risk of human exposure to tick-borne Ehrlichia in the Iberá wetlands ecoregion.

Emerging infectious diseases pose a significant burden on the global economy and public health 1 . The majority of these emerging infectious diseases are zoonoses originating from wild animals 2 . Among arthropod vectors, ticks are responsible for transmitting the greatest diversity of pathogens that affect humans, livestock and companion animals 3 . In the northern hemisphere, Ixodidae transmits the pathogens that cause Lyme disease (Borrelia burgdorferi), human monocytic ehrlichiosis (Ehrlichia chaffeensis) and human babesiosis (Babesia microti). None of these tick-borne parasites is transmitted transovarially; hence, larvae or nymphs must acquire the infection during a blood meal on a infected host [4][5][6] . The probability that a particular host species transmit infection to a feeding vector is often called realized reservoir competence 7 , and it varies between different host species 8 . Since most pathogens can infect multiple hosts, the relative abundance of competent and incompetent host species in a community can cause the abundance of pathogens to vary 9 . The abundance of infected ticks actively seeking a host is affected by both the abundance of ticks in the vegetation and the prevalence of the evaluated pathogen. These factors exhibit significant temporal fluctuations attributed to variations in climatic conditions and changes in the abundance of vertebrate hosts 10,11 . Understanding the complex mechanisms underlying these fluctuations is crucial, as climatic conditions, vertebrate hosts, ticks, and tick-borne microorganisms form intricate biological networks with multiple interactions 12 . Investigating these interdependencies would enhance our comprehension of the fluctuations in the distribution and incidence of tick-borne parasites.
Ehrlichia spp. are intracellular Gram-negative bacteria of medical and veterinary importance that infect monocytes, neutrophils, or endothelial cells, depending on the species involved 4  www.nature.com/scientificreports/ Ehrlichia species have been molecularly detected recently, although their taxonomic positions are still not clearly defined [13][14][15][16][17][18] . The tick Amblyomma dubitatum Neumann is distributed in the biogeographic provinces of Chaco, Pampa, Parana Forest and Atlantic Forest in the Neotropical region of southern South America 19,20 . All stages of A. dubitatum feed predominantly on capybaras (Hydrochoerus hydrochaeris L.) 19,21 . Moreover, immature stages can use small vertebrates as alternative hosts 22 , while all A. dubitatum stages were recorded biting humans 19,23 . In the Iberá wetlands ecoregion (Corrientes province, northeastern Argentina), capybaras are present at high densities, living in close proximity to farms and urban settlements 24,25 . This results in an extensive human-domestic-wildlife interface that may pose a potential risk to public health and animal husbandry.
The aim of this study was to evaluate the infection rate of Ehrlichia species in unfed Amblyomma dubitatum ticks collected from vegetation and investigate its association with capybara density, tick density, and environmental parameters in protected areas within the Iberá wetlands ecoregion in Argentina.

Results
During the twelve sampling sessions, a total of 13,941 ticks were collected from the protected areas studied (Fig. 1). These ticks were identified as A. dubitatum, Rhipicephalus microplus Canestrini, Amblyomma triste Koch, Haemaphysalis juxtakochi Cooley and Amblyomma tigrinum Koch. The total number of tick specimens analyzed individually and in pooled samples, discriminated by species and stage is presented in Table 1.
During the two-year period, the seasonal distribution of questing A. dubitatum exhibited a similar pattern for all stages. Questing larvae and nymphs were collected throughout the entire year. The peak of questing larvae of A. dubitatum occurred in autumn, while nymphs showed peaks in winter and spring. Adult A. dubitatum reached its peak during summer see 26  Two of the 16SrRNA-positive A. dubitatum samples (1 nymph, 1 nymph pool) amplified both the dsb and groEL targets. The 374-bp fragment of the dsb gene obtained from these two samples showed 99.7% identity to the corresponding sequence of Ehrlichia sp. strain San Luis (MH261375) and 97.6% identity to the corresponding sequence of Ehrlichia chaffeensis str. West Paces (CP007480). The 1196-bp fragment of the groEL gene obtained showed 99.9% identity to Ehrlichia cf. chaffeensis from marsh deer (JQ085941) and 98.1% identity to the corresponding sequence of Ehrlichia chaffeensis str. West Paces (CP007480). Phylogenetic analyses using the dsb and groEL sequences from these two samples placed the Ehrlichia sp., hereinafter called Ehrlichia cf. chaffeensis from A. dubitatum, in the same clade as Ehrlichia sp. strain San Luis previously reported to infect A. tigrinum 13 and Amblyomma parvum 27 , as well as Ehrlichia cf. chaffeensis reported in free-ranging marsh deer in Brazil ( Fig. 2A,B). This South American clade of Ehrlichia cf. chaffeensis strains was strongly supported as the sister clade to a clade consisting of several Ehrlichia chaffeensis strains reported in North America ( Fig. 2A,B).   18 . Phylogenetic analysis using these groEL sequences placed the detected ehrlichial agent, hereinafter referred to as "Ca. Ehrlichia hydrochoerus" from A. dubitatum, in the same clade as the novel "Ca. Ehrlichia hydrochoerus" (Fig. 2B) and close to Ehrlichia sp. strain Iberá reported to infect A. tigrinum in the same region 14 .
Statistical analyses were conducted to explore associations between the presence of Ehrlichia in A. dubitatum and host, vector and abiotic variables. The analysis utilized data from both nymphs and adults combined, as there is no evidence of transovarial transmission of Ehrlichia (unfed questing larvae cannot be infected). It is important to note that the number of ticks infected with Ehrlichia cf. chaffeensis from A. dubitatum was insufficient for robust comparisons. Therefore, the statistical analyses were focused solely on ticks infected with "Ca. Ehrlichia hydrochoerus".
Regarding climate variables, the accumulated rainfall in the last 15, 45 and 60 days prior to each sampling (with median values of 27, 63 and 101 mm, respectively) and the average minimum temperature at 45 and 60 days prior to each sampling (with median values of 15.8 and 15.2 °C, respectively) all showed positive effects on the frequency of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum (Fig. 4). The frequency of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum was significantly higher for high values compared to low values of each variable: accumulated precipitation (d -15 : 12.43-fold, p-value = 0.016; d -45 : 3.76-fold, p-value = 0.047; d -60 : 5.43-fold, p-value = 0.011) and average minimum temperature (d -45 : 8.65-fold, p-value = 0.039; d -60 : 5.6-fold, p-value = 0.020). In other words, elevated minimum temperatures and increased accumulative rainfall in the specified time periods were positively associated with the prevalence of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum. No significant associations were observed between "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum and other climatic variables, season, larvae and nymphs abundance.

Discussion
Our findings demonstrate that A. dubitatum in the Iberá wetlands ecoregion is infected by two genetically distinct Ehrlichia species. One of these species is closely related to the zoonotic pathogen Ehrlichia chaffeensis 28 , while the other is similar to the novel "Ca. Ehrlichia hydrochoerus" 18 . Phylogenetic analysis using both the dsb and groEL loci placed Ehrlichia cf. chaffeensis from A. dubitatum in the same clade as Ehrlichia sp. strain San Luis and Ehrlichia cf. chaffeensis from marsh deer, and in proximity to several E. chaffeensis strains from the USA. Further research utilizing additional phylogenetic markers is necessary to determine whether all of these South American ehrlichiae, closely related to E. chaffeensis, are indeed distinct species or constitute an Ehrlichia chaffeensis sensu lato complex, as has been observed for other tick-transmitted rickettsial pathogens in the region 29 .
Using groEL sequences, the other detected ehrlichial agent was identified as "Ca. Ehrlichia hydrochoerus". However, in our study, we were unable to amplify the dsb gene of "Ca. Ehrlichia hydrochoerus" from A. dubitatum. This is consistent with the hypothesis that the commonly targeted dsb sequence is highly polymorphic Table 1. Number of tick specimens analyzed individually and in pooled samples, discriminated by species and stage. *Number of pools/specimens analyzed. The range of specimens per pool is indicated in parentheses. 1 Total number of each species stage ticks. The percentage is indicated in parentheses. In a recent study, we reported the detection of another member of the Anaplasmataceae family closely related to Anaplasma odocoilei in A. dubitatum 26 . To fully understand the potential role of A. dubitatum as a vector of these Anaplasmataceae, further experiments are needed to determine its vector competence. This is particularly important given the zoonotic potential of Ehrlichia cf. chaffeensis and the fact that A. dubitatum is known to parasitize humans in all of its developmental stages 19,23,30 . The capybara is the principal host of all stages of A. dubitatum 19 . In Brazil, two studies were conducted to investigate the presence of ehrlichial agents in capybaras and their associated ticks. In central-western Brazil, Neves et al. 31 reported the absence of ehrlichiae in capybara blood and their associated A. dubitatum. In southern Brazil, Vieira et al. 18 found that "Ca. Ehrlichia hydrochoerus" infects capybara blood but not the salivary glands of A. dubitatum feeding on these capybaras. However, it is important to note that both studies analyzed a small number of A. dubitatum (132 and 11 samples, respectively). In contrast, our study analyzed a large number of ticks from different populations over time, which enabled us to identify the presence of two distinct ehrlichial agents infecting host-seeking A. dubitatum.
Despite capybaras from the same populations in this study being highly infested by A. dubitatum 32 and infected by "Ca. Ehrlichia hydrochoerus" 33 , the prevalence of the ehrlichial agent in the ticks collected from the same site was found to be very low (0.59% of the 2390 A. dubitatum nymphs and adults). Considering the possibility of A. dubitatum having a low susceptibility to "Ca. Ehrlichia hydrochoerus" infection, it is plausible that the bacterial loads present in the capybara's blood might not be sufficient to facilitate infection in the tick. In this scenario, capybaras may play a dual role by acting as key reproductive hosts for A. dubitatum and potentially as incompetent or low realized competent hosts for "Ca. Ehrlichia hydrochoerus". Further research is necessary to test this hypothesis and better understand the specific dynamics between capybaras, A. dubitatum, and "Ca. Ehrlichia hydrochoerus".
Our study revealed that the frequency of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum was not dependent on the density of the tick vector. However, we observed that the likelihood of finding "Ca. Ehrlichia hydrochoerus"-infected A. dubitatum was higher in sites with previously low capybara densities. Since ehrlichiae are not transmitted transovarially 4 , unfed nymphs and adults of A. dubitatum can acquire the infection only through transstadial transmission, which occurs when larvae or nymphs become infected by feeding on a competent reservoir host. Despite A. dubitatum having a one-year life cycle, multiple cohorts can coexist within  www.nature.com/scientificreports/ the same population in the Iberá wetlands ecoregion 26,34 . The pre-moult period for larvae and nymphs of A. dubitatum ranges from 23 to 60 days and 25 to 50 days, respectively, throughout most of the year 34 . Considering that the feeding period for A. dubitatum larvae and nymphs is approximately 6 days 35 , it can be inferred that the majority of larvae and nymphs found in the vegetation during one sampling session will have undergone moulting into nymphs or adults, respectively, two months later (which corresponds to our inter-sampling session interval). Our findings suggest that when the density of capybaras (the primary host of all stages of A. dubitatum) was low in a particular site during the first sampling session (S -1 ), the prevalence of "Ca. Ehrlichia hydrochoerus"-infected A. dubitatum in the same site two months later (S) was higher compared to sites where the density of capybaras was high during the initial sampling session (S -1 ). The immature stages of A. dubitatum in the Iberá wetlands have been reported to feed on various vertebrate hosts in addition to capybaras 22 . Common alternative hosts include the caviine Cavia aperea, the sigmodontines Akodon azarae and Oligoryzomys flavescens, and the marsupial Monodelphis dimidiata 22 . Interestingly, these alternative hosts tend to have smaller home ranges compared to the area where capybara density was estimated [36][37][38] . Based on this observation, we propose a hypothesis that when the density of capybaras decreases in a particular site, the immature stages of A. dubitatum may seek out alternative hosts for blood meals. Consequently, larvae and nymphs of A. dubitatum that feed on these alternative hosts would undergo moulting within the same site, leading to the presence of its nymphs and adults stages questing for hosts in the same site as well. A study on the parasitic Philornis botfly, another host-seeking arthropod parasite, and its multiple bird hosts, demonstrated that the parasite selects alternative hosts only when the principal host is insufficiently available 39 . In the context of our study, it is possible that at least one of the alternative hosts parasitized by immature stages of A. dubitatum exhibits high realized reservoir competence for "Ca. Ehrlichia hydrochoerus", leading to an increased prevalence of this ehrlichiae in questing A. dubitatum nymphs and adults two months later. For Ehrlichia cf. chaffeensis, the low prevalence observed in A. dubitatum prevented any statistical analysis. Nevertheless, considering that capybaras have not been previously reported to be infected by this pathogen, it is plausible that A. dubitatum also acquires Ehrlichia cf. chaffeensis by feeding on alternative hosts. The marsh deer is the only vertebrate in the region known to be infected by Ehrlichia cf. chaffeensis 40,41 . However, it should be noted that the marsh deer is not parasitized by any of the stages of A. dubitatum 19,21 . Co-feeding transmission of Ehrlichia muris-like agent was demonstrated for Ixodes scapularis larvae feeding along with infected-nymphs 42 . Thus, a tick previously fed on an infected marsh deer could transmit Ehrlichia cf. chaffeensis by co-feeding to A. dubitatum in the same capybara host. In the Iberá wetlands ecoregion, A. triste is the only tick species reported to parasitize both marsh deer and capybaras 22,32 . However, A. triste parasitism of capybaras is infrequent 32 and this tick species solely feeds on marsh deer during the adult stage 19,21 . In addition, it is important to consider that other large mammals (such as Sus scrofa, Axis axis, Lepus europaeus, Myrmecophaga tridactyla) as well as birds are also utilized as alternative hosts by the immature stages of A. dubitatum, albeit to a lesser extent 19,21,22 . Therefore, the possibility that these hosts may serve as competent reservoir for "Ca. Ehrlichia hydrochoerus" and/or Ehrlichia cf. chaffeensis should not be disregarded.
Regarding environmental variables, we showed a positive correlation between high minimum temperatures and previously cumulated rainfall with the prevalence of "Ca. Ehrlichia hydrochoerus" in A. dubitatum. Abiotic factors not only have direct effects on tick fitness but may also modulate pathogen development and growth in ticks [43][44][45] . Parasites are not harmless to their hosts, and this holds true for tick-borne rickettsial pathogens as well, which can cause a decrease in both tick survival 46 and moulting success 47 . Given that the prevalence of "Ca. Ehrlichia hydrochoerus" does not depend on A. dubitatum density, it can be hypothesized that the observed positive relationship may be attributed to the increased survival of "Ca. Ehrlichia hydrochoerus"-infected A. dubitatum under favorable conditions. Possible explanations for these observations include heightened tolerance of the infection in hosts that are in good condition 48 or temperature-driven changes in the transcriptional profile of the bacteria that affect virulence 49 . To test these hypotheses, additional field and laboratory experiments are required.
In summary, our findings suggest that a combination of biological factors such as capybara density, along with abiotic factors including temperature and accumulated precipitation, may contribute to an elevated risk of human exposure to tick-borne Ehrlichia in the Iberá wetlands ecoregion.

Methods
Study area. The study was conducted in the Iberá wetlands ecoregion, which encompasses a system of estuaries, baths, shallow lakes, and watercourses interconnected within an area of approximately 40.415 km 2 . The primary source of water in the wetlands is rainfall, with an average historical precipitation of 1700-1800 mm. During the summer, rainfall is slightly higher (600-700 mm) compared to other seasons. The climate in the region is humid and subtropical. The monthly average minimum temperature in June and July is around 16 °C and 17 °C, respectively, while the average maximum temperature occurs in January and February, ranging between 27 °C and 28 °C.
The questing ticks used in this study were collected from two protected areas known to have large populations of capybaras 25  www.nature.com/scientificreports/ 10 min during this period. In addition, drag sampling was conducted in three parallel 100 m transects, located outside the transect where the CO 2 traps were placed. During dragging, a 1 × 1.5 m cloth was dragged along the ground, and ticks were collected by inspecting the cloth approximately every 5 m. The tick collection surveys were carried out during the mid-morning and mid-afternoon periods, specifically avoiding the hottest hours of midday.
The taxonomic determination of larvae, nymphs and adult ticks collected was conducted following Joan 50 , Guglielmone and Viñabal 51 and Nava et al. 19 , and by comparison with known laboratory-reared specimens deposited in the tick collection of INTA Rafaela, Argentina. Then, all ticks were processed for DNA extraction by a boiling technique 52 . Adult ticks were processed individually, while nymphs and larvae were processed either individually or in pools. The pooling of nymphs and larvae was based on date, trap, species and abundance.
All samples were screened for Ehrlichia infection using a previously described real-time PCR assay that targets the 16SrRNA gene 27 . This assay is capable of identifying the genus involved through melting curve analysis 26 .
Positive samples were further tested by amplifying the dsb and groEL genes, as described 53,54 . The integrity of DNA obtained from ticks was checked using primers that amplify a portion of the arthropod 16SrRNA 55 , as previously described 52 . For all PCR reactions, positive controls (Ehrlichia canis or Amblyomma triste) and negative controls (molecular-grade water) were included. The resulting PCR products were sequenced directly using amplifying primers. Phylogenetic analyses were conducted using the Maximum-likelihood (ML) method with MEGA 7.0.
To estimate the relative density of capybaras at each site (number of capybaras per kilometer of shoreline), we utilized the frequency of fresh capybara faeces (pellet groups) observed along the shoreline. This indirect method, known as pellet group count, is a reliable indicator of capybara presence 56 . Alongside tick sampling, transects parallel to the shoreline were examined for the presence of fresh capybara pellet groups. For National Park Mburucuyá, two 300-m-long transects were used, while for Laguna Iberá and Rincón del Socorro, three 100-m-long transects were used. The transects were 5 m wide and separated by 200 m from each other. Transects were established based on the terrain limitations such as the shape of the water body and the presence of dense vegetation. The estimation of capybara density from pellet group counts was done using the modified Eberhard and Van Etten 57 model. The capybara defecation rate used in the calculation was 4.4 faeces per individual per day, which was estimated from a previous study conducted in the same area 56 .
To estimate the abundance of A. dubitatum present in the study areas, the total number of ticks collected was counted and categorized by stage, season and site. The climatic variables recorded were cumulative precipitation and average minimum, mean and maximum temperature (meteorological records were obtained from: https:// centr ales. bolsa cer. org. ar/). Statistical analysis. The response variable was the presence or absence of Ehrlichia DNA in ticks, a dichotomous variable. The independent variables used included larvae and nymph abundance, capybara density, site, season (southern hemisphere), precipitation levels and temperature. All variables were transformed into dichotomous variables by dividing the values into high and low categories based on the median value. Variables that incorporated time delays (time lags) were analyzed by considering the number of previous samplings (indicated as S) or days (indicated as d), depending on the specific variable being analyzed. The variables "larvae abundance" and "nymph abundance" were evaluated at a time lag S -1 , while "capybara density" was evaluated at time lags S -1 and S -2 , representing approximately 60 and 120 days, respectively. The variables "temperature" and "precipitation level" were evaluated using time lags of d -15 , d -30 , d -45 and d -60 . Additionally, the time lag ranges d -15,-30 ; d -30,-45 and d -45,-60 were considered. For the statistical analysis, all DNA tick samples were treated as pools, while individually processed ticks were considered as a pool with size equal to 1.
The frequency of Ehrlichia presence in ticks was expressed as the Minimum Infection Rate (MIR), defined as the lower limit of the true infection rate. The MIR was calculated as the ratio of the number of positive pools to the total number of ticks tested. The MIR assumes that only one infected individual exists in a positive pool 58 . The presence of Erhlichia in pool ticks samples was adjusted for the size of each pool and analyzed using logistic regression models. All statistical analyses were performed using the PoolTestR package 59 in R version 4.2.1 60 .

Data availability
The sequences generated and analysed during the current study are available in the GenBank repository, accession numbers OR001765-OR001775.